L4: Nucleic Acid Structure and Detection

Question 1

What is a nucleic acid strucutre

Answer 1

1. nitrogenous base unit: purine of pyrimidine
2. 5B sugar unit: ribose or deoxyribose
3. phosphate group

Question 2

chargaff’s Rule

Answer 2

total Purine (AG) = total prymidine (TC)

Question 3

Who predicted the d helix strucutre

Answer 3

• Watson and Crick, complementary bases held together by hydrogen bonds

Question 4

How does base pairing work in DNA

Answer 4

• 2 hydrogen bonds for AT
• 3 hydrogen bonds for GC
• sugar/phosphate backbones are antiparallel ( 3’aligned with 5’)

Question 5

What are the three d helix conformations

Answer 5

A- DNA short and wide right handed
B-DNA common
Z- left handed

Question 6

Briefly go through chromosomes

Answer 6

• genome of species
  human: 22+ X/Y
  barley = 7
  homologous chromomes: 2n or 1n cells
  structure: nucelosomes (proteins + DNA), arms, centromere, telomere

Question 7

One chromosome =

Answer 7

one DNA molecule

Question 8

What are the elements of nuclear gene

Answer 8

1. Distal Regulatory
2. proximon elements
   = promoter region

Transcriptional start sitre

Exon + Intron + Exon
= structural region

Transciptional termination site

Question 9

Define Intron and Exon

Answer 9

Intron: intervening sections of non coding sequencing removed during processing of primary transcript

Exon: sequence from primary transcript, coding

Question 10

What are restriction enzymes / endonucleases

Answer 10

• recognise specific nucleotides and produce a cut in ds DNA
• recongise 4-8 nucleotides usually 4pb, 6 is medium and 8 is rare
• palindormic

Question 11

Name the applications
T4 DNA ligase
T4 RNA ligase
Taq Poly
Phosphatase
T4 Polynucloetide Kinase
DNAse 1
MMLV Reverse
AMV Reverse
Proteinase K
Ribonuclease A
Nuclease S1
Mung Bean Nuc

Answer 11

T4 DNA Ligase: cloning

T4 RNA ligase: RNA labeling, primer extension

Taq DNA Polymerase: PCR from DNA template
Phosphatase: prevent recircularization of DNA vectors
T4 Polynucleotide Kinase: Nucleic acid labeling
DNase I: elimination of DNA in RNA or protein
preparations

M-MLV Reverse Transcriptase: RT-PCR for long transcripts

AMV-Reverse Transcriptase Synthesis: of cDNA from RNA (RT-PCR)

Proteinase K: Removes proteins from DNA samples

Ribonuclease A: RNA elimination from DNA preparation

Nuclease S1: Degrades RNA or single-stranded DNA into 5′ mononucleotides

Mung Bean Nuclease: Degrades single stranded DNA and RNA

Question 12

Difference between EcoRI and EcoRV

Answer 12

• EcoRI: produces sticky ends
• EcoRV: produces blunt ends

Question 13

Name two blotting techniques and define them

Answer 13

Southern Hybridisation: binding radioactive/fluro DNA probe to DNA molecule immobilized on membrane filter (DNA by DNA)

Northern Hybridisation: “”” DNA probe between complementary bases in RNA and DNA probe

Question 14

How do you determine the optimum temperature for the probe

Answer 14

Tm = 4(G+C) + 2(A+T) assumed 0.9M

Question 15

What is UV Absorabce Quanitifaction based on

Answer 15

sample aborbance of light wat wavelength 260, due to aromatic nitrogen bases, related to nucleic acids

Question 16

What are the general approximation of UV absorbance quantification

Answer 16

If A260
1 ds DNA = 60
ssDNA = 40
oligo = 30
equi molar amounts: 2:1

Question 17

How does detection take place + details

Answer 17

visualisation of NA after seperations
- use small amounts of DNA/RNA but need good estimate of amount of DNA/RNA
use dyes like EtBr, prop Idone, SYBR green, RedSafe

Question 18

How does gel electrophoreisis work

Answer 18

1. seperate by length and size
2. apply electric field and neg molecules move through argose
3. short molecules travel farther
4. proteins seperated by charge becaus eof the pores are too big to sieve proteins